This grant represents a systematic study of a novel antiviral strategy called capsid-targeted viral inactivation (CTVI). In this strategy, a viral protein is attached to a deleterious enzyme,such as a nuclease. The virus-nuclease fusion protein is targeted to assembling virus particles intracellularly, and then destroys the virus particle after it has assembled. The figure depicts the CTVI strategy for an RNA-Virus and a capsid protein-RNAse fusion: a) Because viral capsid proteins self- assemble, such deleterious fusion proteins can co-assemble with wild-type capsid proteins into the virion; b) the nucleic acid within the hybrid virion carrying the interfering fusion protein can then be hydrolyzed by the nuclease. CTVI represents a novel and potent antiviral method that may eventually be applied to viral diseases of animals and humans. Antiviral fusion genes could eventually be expressed in transgenic animals, conferring virus resistance upon them, or they could be introduced into infected individuals by gene therapy. We will study the assembly of retroviral particles, and particularly with regard to the behavior of fusion proteins consisting of capsid protein and "reporter protein" moieties. Information obtained from these studies will expand the understanding of basic assembly processes used by retroviruses and the related yeast element Tyl. Retroviruses are attractive for study because they cause disease, much is known about the assembly, process, and they represent true infectious agents. The Tyl system brings complementary strengths: ease of genetic manipulation, and an envelope-free cytoplasmic viral assembly process. We will continue our studies of CTVI reported in the Tyl system and expand our analysis of CTVI in murine retroviruses. The specific aims of this proposal include experiments designed to understand the basic parameters defining assembly of fusion proteins into Tyl and retrovirus virions, and optimization studies designed to empirically determine the structures of the most potent antiviral fusions. A major emphasis will be placed on extensive characterization of antiviral effects we have observed in preliminary experiments with Moloney murine leukemia virus.